Biofilms that colonize tooth surfaces and epithelial cells lining the periodontal pocket/gingival sulcus (subgingival dental plaques) are among the most complex biofilms that exist in nature and lead to periodontal diseases. Our long-term goal is to develop a clinically appropriate way to enhance the penetration and effectiveness of photoactive compounds (or photosensitizers) in human dental plaque for prevention, control and/or treatment of periodontitis. The hypotheses to be tested are: a) Methylene blue (MB)-loaded polymeric nanoparticles can fully penetrate oral multispecies biofilms evolved from natural human dental plaque in vitro, and b) Complete photodestruction of these microbial biofilms in vitro may be possible after their sensitization with MB-loaded nanoparticles followed by exposure to red light of 665 nm. The specific aims of this R21 proposal are: 1. To prepare and characterize MB-loaded biocompatible and biodegradable polymeric nanoparticles. We will (i) prepare MB-encapsulated nanoparticles of poly(lactic-co-glycolic acid) with anionic and positive surface charge, (ii) evaluate their size, surface charge, and morphology by ZetaPALS instrument and scanning electron microscopy, (iii) verify their capacity and efficiency of MB encapsulation by spectroscopy, and (iv) investigate the in vitro release of MB in simulated environment to insure that sufficient agent is available for effective photodynamic therapy. 2. To investigate the distribution of MB-loaded nanoparticles in biofilms and their phototoxicity on biofilm microorganisms in vitro. We (i) develop multi-species microcosm laboratory biofilms in vitro using saliva and human dental plaque, (ii) investigate the internalization of MB-loaded nanoparticles by bacteria using transmission electron microscopy, (iii) assess their penetration into the biofilms by confocal scanning laser microscopy, and (iv) evaluate their photodynamic effects on biofilm species by total viable counts, an species identification using DNA probes and PCR. PUBLIC HEALTH RELEVANCE: This project is an attempt to target dental plaque bacteria using visible light and microscopic particles loaded with a photoactive drug. Our research may help develop photodynamic therapy as a non-invasive technique for prevention, control and/or treatment of periodontal disease.